ELSEVIER

Life Seicnccs, Vat. 64, No. 21, pp. 1897-1915 1999 copyright 0 1999 elsevier scicncc Inc.

Printed in the USA. All rights raervcd 0024-3205/99/S-e front matter

PI1 SOO24.3205(99)00136-S

INTRAGASTRIC AND INTRAPERITONEAL ADMINISTRATION OF CrylAc PROTOXIN

FROM BACILLUS THURINGIENSIS INDUCES SYSTEMIC AND MUCOSAL ANTIBODY

RESPONSES IN MICE

Roberto I. Vazquez-Padron , Leticia Moreno-Fierros', Leticia Neri-

Baz6n3, Gustav0 A. de la Rival and Ruben Lopez-Revilla3

'.Center for Genetic Engineering and Biotechnology (CIGB) P.O. Box

6162, 10600, HAVANA, CUBA; ' ENEP-IZTACALA-UNAM, P.O. Box 314,

Tlalnepantla, ED0 MEXICO, MEXICO; 3 Department of Cell Biology,

CINVESTAV-IPN; P.O. Box 14-740, 07000 MEXICO D.F.

(Received in final form February 9, 1999)

Summary

The spore-forming soil bacterium Bacillus thuringiensis

produces parasporal inclusion bodies composed by 6-

endotoxins also known as Cry proteins, whose resistance to

proteolysis, stability in highly alkaline pH and innocuity

to vertebrates make them an interesting candidate to

carrier of relevant epitopes in vaccines. The purpose of this study was to determine the mucosal and systemic immunogenicity in mice of CrylAc protoxin from B. thuringiensis HD73. Crystalline and soluble forms of the

protoxin were administered by intraperitoneal or intragastric route and anti-CryIAc antibodies of the major

isotypes were determined in serum and intestinal fluids.

The two forms of CrylAc protoxin administered by

intraperitoneal route induced a high systemic antibody

response, however, only soluble CrylAc induced a mucosal

response via intragastric. Serum antibody levels were

higher than those induced by cholera toxin. Systemic immune

responses were attained with doses of soluble CrylAc

ranging from 0.1 to 100 pg by both routes, and the maximal

effect was obtained with the highest doses. High anti- CryIAc IgG antibody levels were detected in the large and

small intestine fluids from mice receiving the antigen via

IP. These data indicate that CrylAc is a potent systemic

and mucosal immunogen.

Key WO&: CrylA proteins, Bacillus thuriqiensis, intestinal immunity, antibody response

Address for correspondence: Roberto I. Vazquez-Padron Center for

Genetic Engineering and Biotechnology (CIGB) P.O. Box 6162, 10600, HAVANA, CUBA. Tel: (53) 7 216022/ 218466; Fax: (53) 7 218070/336008; email: Jrriva@ciRb.edu.cu

1898 Immunogenicity of CrylAc Protoxin Vol. 64, No. 21, 1999

Bacillus thuringiensis is the major bacterial species used as bioinsecticide. During sporulation, bacterial cells produce insecticidal inclusion bodies formed by proteins (Cry proteins)

active against larvae of invertebrate species belonging to insects, nematodes and protozoa (1). Biochemical properties of Cry

proteins such as high resistance to proteolysis, solubility and

stability in highly alkaline pH, as well as a demonstrated

innocuity to vertebrates (2) make them an interesting alternative

for the development of carriers of relevant epitopes in vaccines.

However, there are a very few studies on the physiological or

immunological effects of the Cry protein family on vertebrate

organisms, despite the known homology of B.thuringiensis with the

pathogenic B. cereus species (3).

When Cry proteins are used to immunize mice or rabbits following

the conventional protocols for parenteral immunization that

include Freund's adjuvant, high antibody titers are induced (4).

An old report indicates that crystalline Cry proteins have anti-

tumor activity against Yoshida ascites sarcoma possibly due to

their ability to enhance general immunity in rats (5). The same

authors have demonstrated the enhancement of mice immune response

to sheep red blood cells by B. thuringiensis insecticidal crystals

(6). On the other hand, high concentrations of B. thuringiensis

spores and crystals from several B. thuringiensis strains have

shown no toxicity to vertebrates (3).

The present study was performed to examine the mucosal and

systemic immunogenicity in mice of CrylAc protoxin from B.

thuringiensis subsp. kurstaki HD73. Crystalline (cCrylAc) and

soluble (sCrylAc) forms of the antigen were administered to mice

by intraperitoneal (IP) or intragastric (IG) route and anti-CrylAc

antibody responses were determined. CryIAc administered in

microgram amounts by both routes induced an intense systemic

antibody response as well as the secretion of specific mucosal

antibodies. Our results support the possibility of using CryIAc

protoxin as a carrier antigen in oral or parenteral vaccination.

Methods

Organisms and culture conditions

B. thuringiensis var kurstaki HD73 was supplied by the Center of

Genetic Engineering and Biotechnology (Havana, Cuba) and grown in

liquid GR medium (10 g/l glucose, 3.20 g/l peptone, 3.20 g/l yeast

extract, 1.53 g/l NaH,PO,, 1.85 g/l Na2HP0,, 0.5 g/l KCl, 0.3 g/l MgS0,7H,O, 0.1 g/l CaC1,2H,O, 0.075 g/l Ferric citrate, 0.05 g/l MnSO,, 0.0075 g/l ZnS0,7H,O, 0.0045 g/l CuSO,) (7). Escherichia coli JM103 (pOS9300) was gently provided by Dr. Donald H. Dean, Ohio State University, COLUMBUS (USA). Induction of CrylAc protein

expression was performed in liquid LB medium containing 50 pg ofm

ampicillin per ml using isopropyl P-D-thiogalactopyranoside (IPTG)

(7).

Vol. 64, No. 21, 1999 Immunogenicity of CrylAc Protoxin 1899

Xmmunogens

Cholera toxin (CT) and bovine serum albumin (BSA) were purchased

from Sigma Chemical Co. (St. Louis, MO). Crystalline CrylAc was

purified from sporulated B. thuringiensis cultures (9). Briefly,

crystals and spores were harvested by centrifuging at 6,000 g for

10 min, washed twice with ice-cold 1M NaCl and resuspended in 0.5

ml of 0.1% Triton X-100 solution. The mixture was loaded onto a 12

ml 70-85% (w/v) sucrose gradient and centrifuged at 40,000 g for

1h at 4°C. The crystal band was collected with a syringe, diluted

with cold water and then pure crystals were harvested by

centrifugation at 10,000 g for 15 min. The crystals were

resuspended in distilled water and freeze-dried.

Soluble CrylAc was purified from IPTG-induced E. coli JM103

(pOS9300) cultures (7). The cell pellet harvested by

centrifugation was resuspended in TE buffer (50 mM Tris-HCl pH 8,

50 mM EDTA) and sonicated (Fisher Sonic Dismembrator Model 300)

three times for 5 min in ice. Inclusion bodies were collected by

centrifugation at 10,000 g for 10 min. The pellets were washed

twice with TE buffer, solubilized in CBP buffer (0.1 M Na,CO, pH

9.6, 1% P-mercaptoethanol, 1 mM PMSF) and particulate material was

discarded by centrifugation. Purified proteins were examined by

SDS-PAGE (10) and protein concentration was determined using the

Bradford's method (11).

Immunizations

In all experiments, female 8-10 weeks Balb/c mice were used.

Immunization was carried out according to Coligan et al. (12). The

antigens were administered via IP in 0.1 ml phosphate buffered

saline (PBS), or via IG in 0.1 ml magnesium-aluminum hydroxide

suspension (Maalox). Experimental groups were formed by five

female mice each one to which three antigen doses were applied on

days 0, 7 and 14. Mice were sacrificed 7 days after the last

immunization. The experiment performed to determine the immunogenicity of CrylAc protoxin required 12 groups of mice. The

immunogens administered via IG or IP were: 1) 100 pg cCrylAc, 2)

100 pg sCrylAc, 3) 100 pg cCrylAc plus 10 pg CT, 4) 100 pg sCrylAc

plus 10 pg CT and 5) 100 pg BSA plus 10 pg CT. Control mice

received 100 pg BSA alone. The immunogens were applied individually

using only one immunization route. In this experiment, the antibody response was measured in feces and serum.

The experiment performed to determine the dose-response relation

for sCrylAc applied through both immunizations routes, eight experimental groups were immunized with the following four doses:

0.1, 1, 10 and 100 pg per mouse. Mice were sacrificed on day 21 and serum and large and small intestine fluids were collected.

1900 Immunogenkity of CrylAc Protoxin Vol. 64, No. 21, 1999

Sample collection

Fresh feces were harvested from live mice and pooled by groups

(13). Subsequently, 1 g of feces was resuspended in 600 ~'1 of ice-

cold PBSM buffer (5% non-fat milk in PBS) containing 100 mM of p-

hydroxy-mercuribenzoic acid (pHMB) (SIGMA, St. Louis, MO) , particulate material was discarded by centrifugation and

supernatants were stored at -20°C. Serum samples were obtained from

blood extracted by cardiac puncture of ether-anesthetized mice.

Contents from small and large intestines were collected by the

method described by Moreno-Fierros et al (14). Contents from the

small and large intestines were flushed out with 5 ml and 3 ml of

cold PBSM, respectively. The fluid was supplemented with 100 mM of

pHMB and centrifuged for 10 min at 8,000 g. The supernatants were

frozen immediately in liquid nitrogen and stored at -20°C.

ELISA

Antibody levels in sera and intestinal fluid were determined by an

enzyme-linked immunosorbent assay (12). Briefly, 96-well plates

were coated with 100 ~1 of sCrylAc (10 pg/ml) or CT (5 pg/ml) in

carbonate buffer pH 9.6. Plates were incubated 2h at 37'C and

washed three times with 0.05% Tween 20 in PBS buffer (PBST).

Blocking was performed with PBSMT (1% nonfat dry milk in PBST).

Further washing was done with PBST. Serial dilution of sera and

fecal supernatants was done with PBSMT. Volumes of 100 ~1 from

small and large intestinal fluids were added to the micro wells.

The plates were incubated overnight at 4OC, washed with PBST and

anti-IgG (Pierce, Rockford, IL), anti-IgM (Pierce Rockford, IL) or

anti-IgA (SIGMA, St Louis, MO) secondary antibodies (peroxidase-

labeled goat anti-mouse) were added at room temperature for 2h.

The plates were washed and the enzymatic reaction was developed

with substrate solution (0.5 mg/ml o-phenylendiamine, 0.01% H,O, in

0.05 M citrate buffer pH 5.2). Within 15 min, the reactions were

stopped with 2.5 N H,SO, and the absorbance at 492 nm (A,,,) was

measured using an ELISA Multiskan reader (Anthos Labtec

instruments, USA). The background was established as the dilution

of serum or intestinal fluid from control mice with the highest

A 492 * Titers were defined as the reciprocal of the highest endpoint

sample dilution with an A,,, value 0.1 higher than the background

value. The anti-CrylAc or anti-CT antibody levels in non-immunized

mice were as similar as those in the control at the end of each

experiment. Specific antibody levels in intestinal fluids were

expressed as the corresponding Aag, values.

Calculations and statistics Antibody levels were converted to logarithms for calculation of

means, standard deviation and rank. The significance of

differences between groups was tested using the Mann-Whitney test

and the differences noted by the Newman-Keuls test (15).

Vol. 64, No. 21,lW Immunogeoicity of CrylAc Protoxin 1901

Results

Immunogenicity of crystalline and soluble CrylAc

To test CryIAc immunogenicity, antigen doses of 100 pg were

ad:.inistered to Balb/c mice by IP and IG routes. Groups of five

animals each one were injected three times either with the cCrylAc

or the sCryIAc protoxin. In additional groups of animals CT was

co-administered as an adjuvant with both forms of the protoxin.

By IP immunization, the protoxin alone or together with CT induced

the highest titers of serum anti-CrylAc IgG and IgM antibodies.

The log-titers induced by sCrylAc were 4.53 for IgM and 6.41 for

IgG. The IgG antibody response induced by SCrylAc was about lo-

fold higher than that induced by cCrylAc. The IgM response was

twice higher with the soluble form than with the crystalline form

of the protoxin. In contrast with sCrylAc, crystalline CrylAc did

not induce specific IgA antibodies by this route (Fig I).

IP co-administration of cCrylAc with CT increased anti-CrylA IgM

an ibody levels. CT had no effect on the IgM antibody response

when it was co-administered with sCrylAc. IgA antibody responses

were elicited when cCrylAc was co-administered with CT by the IP

route.

Like by IP route, high IgM and IgG titers were obtained when the

protoxin was administered by IG route, showing log-titers of 2.70

and 5.17, respectively. Soluble CrylAc induced an IgG antibody

response about 10 times higher than that of cCrylAc, while the IgM

response attained with the latter was on the contrary five times

higher than with the soluble form of the protoxin. In contrast

with the IP route, cCrylAc induced an serum IgA antibody response

by IG route higher than that of SCrylAc (Fig 1).

IG co-administration of sCrylAc with CT had no effect on the level

of serum IgG antibodies, whereas IgM antibody production was

stimulated. CT co-administered with cCrylAc increased the IgG antibody response but not that of IgM antibodies. The IgA antibody

response to both forms of the protoxin was not significantly affected when co-administered with CT. As expected, immunization

with BSA via IP and IG did not induce detectable serum anti-.CrylAc

antibodies (Fig 1).

Anti-CrylAc antibodies from feces

Intestinal antibody levels induced by CrylAc immunization were estimated in feces.

alone or with CT, IP immunization with cCrylAc or sCrylAc either

Using the IG route, induced anti-CrylA IgG and IgA coproantibodies.

anti-CrylAc IgG and IgA responses were induced

1902

cCrylAc

cCrylAc-CT

sCrylAc

sCrylAc-CT

cCrylAc

cCrylAc-CT

sCrylAc

sCrylAc-CT

cCrylAc

cCrylAc-CT

sCrylAc

sCrylAc-CT

Immunogeoicity of CrylAc Protoxin

b a IgA 1

a a

a

a a

Vol. 64, No. 21, 1999

a 7 654321 2 3 4 5 6

Anti-CrylAc antibody titers a

Fig 1.

Serum anti-CrylAc antibody responses in mice seven days

after the last immunization with 100 pg of cCrylAc or

sCrylAc alone or with 10 pg of CT. The antigens were

administered by IP or IG route. The IgM, IgG and IgA

antibody log-titers were determined by ELISA. Control

groups, mice immunized with BSA alone, showed serum

antibody log-titers cl. Bars represent the means of log-

titers f standard deviation of each experimental group with

n=5. The letter on the bars represents the differences

noted by the Newman-Keuls test (p<O.Ol)

only with sCrylAc; in this case co-administration with CT did not

change the magnitude of the mucosal IgA response. However, IgA and

IgG antibody titers increased significantly when cCrylAc was co-

administered with CT by the IG route. The IgG antibody responses by IP immunization were higher than those induced by the IG route.

In contrast, the IgA antibody responses induced by IG immunization

with sCrylAc alone or with cCrylAc plus CT were higher than those

attained using the IP route (Fig 2).

Vol. 64, No. 21, 1999

cCrylAc

cCrylAc-CT

sCrylAc

sCrylAc-CT

cCrylAc

cCrylAc-CT

sCrylAc

sCrylAc-CT

Immunogenicity of CrylAc Protoxin 1903

I , I I I

4 3 2 1 2 3 4

Anti-CrylAc coproantibody titers

Fig 2.

Anti-CrylAc antibody responses in feces. Groups of mice

(n=5) were immunized three times using 100 ug of either

cCrylAc or SCrylAc form alone or with 10 pg of CT. The

antigens were administered by IP or IG route. Feces from mice were collected, pooled by groups and the IgM, IgG and

IgA titers were determined by ELISA. Pooled feces from mice

immunized with BSA alone via IP or IG, had a coproantibody

log-titers ~1. Bars represent the log of titer. IgM antibodies were not found in the samples.

The adjuvant effect of CT on the anti-CrylA mucosal immune

response, inferred from the increase of specific IgA and I.gG antibody levels, was only observed after IG immunization with

cCrylAc. The IgA responses increased slightly when CT was co-

administered via IP with both protoxin forms, whereas IG administration of sCrylAc alone yielded similar results. Anti-

CrylAc IgM antibodies were not detected in feces.

Anti-CT serum and fecal antibodies

We also analyzed the anti-CT antibodies in sera and feces of mice

to which CT was co-administered with cCrylAc, sCrylAc or BSA.

1904 Immunogenic&y of CrylAc Protoxin Vol. 64, No. 21,1999

Serum anti-CT IgM antibodies were produced after IP coadministration of CT with BSA, CCrylAc or sCrylAc. Anti-CT IgM antibodies were elicited only by IG co-administration of CT with

sCrylAc. IP immunization of CT with sCrylAc elicited serum anti-CT

IgG antibodies, however, the same effect was observed via IG only when CT was administered with cCrylAc (Table I).

Anti-CT IgG and IgA coproantibodies were detected after IP or IG

immunization using CT co-administered with BSA, cCrylAc, or sCrylAc. The strongest anti-CT IgG coproantibody response was

produced when CT was coadministered with BSA by both routes. When

CT was co-administered with cCrylAc or sCrylAc by IP route a

stronger anti-CT IgG coproantibody responses was induced compared

to those by IG route (Table II).

Anti-CT IgG fecal antibody titers were higher than those of IgA in

all cases. The magnitude of the anti-CT IgA antibody response was

significantly higher when CT was coadministered with cCrylAc than

with BSA or sCrylAc. In contrast, the IgG anti-CT coproantibody response when CT was coadministered with BSA or sCrylAc was higher

than with cCrylAc.

The magnitude of the anti-CT IgA and IgG coproantibody responses

was similar after IG co-administration of CT with both protoxins

forms. Anti-CT IgG coproantibody titers were higher than the IgA

titers only when CT was co-administered with BSA using the IG

route. IG co-administration of CT with CrylAc had no influence on

the magnitude of the anti-CT IgA responses.

Dose effect on sCrylAc imunogenicity

The dose-response experiments were performed with sCrylAc because

it was able to induce both serum and corporal antibody responses

without CT as adjuvant. All four doses of sCrylAc (0.1, 1.0, 10

and 100 pg) elicited higher levels of serum anti-CrylAc antibodies

when the IP route rather than the IG route was used. The maximal

effect was attained with 100 pg. The serum anti-CrylA IgG antibody

titers depended on the antigen dose, whereas the IgM and IgA serum

antibody responses induced via IP were similar for all doses (Fig

3).

The serum anti-CrylA IgG antibody titers attained by IG route were

lower than those obtained by IP route. The magnitude of IgG, IgA

and IgM serum anti-CrylA antibody responses produced after IG

immunization was dose-dependent between 0.1 and 10 pg.

The large intestine fluid anti-CrylAc antibody levels elicited by

different doses of CrylAc administered by both routes are shown in

Fig 4. In the large intestine fluid, high anti-CrylAc IgG antibody

levels were detected after administration of all doses via IP,

Vol. 64, No. 21, 1999 Immmmgenicity of CrylAc Protoxin

Table I

Serum antibodies induced in mice by CT.

IR’ lmmunogen CT

Arithmetic mean of serum antibody titer (range)’

IgA IgG tgM

cCrylAc + 3.90a (3.60, 4.08) 5.01 b (4.88, 5.10) 4Z%la (4.10, 4.53)

IP sCrylAc + 2.47b (2.23, 2.70) 6.24’ (5.17, 6.53) 4.20’ (4.10,4.32)

BSA + 3.70’ (3.51,3.90) 4.94 b(4.74, 5.08) 3.87 b (cl, 4.08)

sCrylAc - cl cl cl

cCrylAc + 3.50b (3.39,3.68) 5.20a (4.26, 5.26) <l

IG sCrylAc + 3.2Qb (3.10, 3.45) 3.21 b (4.70, 4.88) 3.24 (-1, 4.03)

BSA + 4.80a (4.62, 4.95) 4.80a (4.60,4.92) <l

sCrylAc - <l cl <l

1905

' Immunization route: CrylAc or BSA were administered alone or

with CT via IG or IP. 2 The IgA, IgG and IgM antibody titers were measured by ELISA.

Sera from mice immunized with sCrylAc alone were used as control to establish the background values. Significant differences within each group are indicated with a letter (P < 0.05; Newman-Keuls

test).

Table II

Coproantibodies induced in mice by CT.

IR’ lmmunogen CT

Arithmetic mean of coproantibody titers!

tgA tgG tgM

IP cCrylAc + 1.95 2.04 <l sCry1 AC + 1.30 2.78 <l

BSA + 1.60 3.00 <l sCrylAc - <l cl <l

cCrylAc + 1.70 1.60 <l IG sCrylAc + 1.88 1.93 <I

BSA + 1.60 3.00 1c sCrylAc - <l <l <I

’ Immunization route: CrylAc or BSA were administered alone or with CT using IG or IP route. ’ Feces were collected, pooled by groups and specific IgA. IgG and IgM coproantibody titers were measured by ELISA. Pooled feces from from mice immunized with sCrylAc alone were used as control to establish the background values.

being 100 c(g the dose inducing the highest IgG and IgM antibody

titers. The intestinal anti-CrylAc IgG response was dose- dependent, except for 1 pg of the antigen, which had an effect

slighter than that of 0.1 pg. In contrast with the serum antibody responses, no IgA coproantibodies were detected in the large intestinal fluids. After IG immunization, production of anti- sCrylAc IgG coproantibodies was detected only with doses of IO and

100 pg.

lw6 Immunogenicity of CrylAc Protoxin Vol. 64, No. 21, 1999

IgM

IgG

IgA

IP IG

6 5 4 3 2 1 2 3 4 5 6

Anti-CrylAc serum antibody titers

Fig 3

Serum antibody responses to several doses of sCrylAc. The

antigen was administered by IP or IG route. The doses used

were 0.1, 1, 10 or 100 pg of CrylAc diluted in PBS or

Maalox. The IgM, IgG and IgA antibody titers were

determined by ELISA. The antibody log-titers in serum from

control mice were cl. Bars represent the means of log

titers f standard deviation of each experimental group

(n=5).

Vol. 64, No. 21, 1999 Immunogenicity of CrylAc Protoxin 1907

IP IG

IgM

1gG

'gA

1,6 1,2 0,8 0,4 0,O 014 0.8 '32 ft6

A 492

Fig 4

Anti-CrylAc antibody secretions in large intestine induced

by several doses of CrylAc. The antigen (0.1, 1, 10, 100 1.19

of CrylAc) was administered by IP or IG route. To collect

the intestinal contents, immune mice were sacrificed and the large intestine flushed out with 3 ml of cold PBSM

buffer. The IgA, IgG and IgM coproantibody levels were

measured by ELISA. The mean of A,,, values of control mice

were 0.085 k 0.013. Bars represent the level of antibodies expressed in arbitrary units of A,,, _ + standard deviation of each experimental group (n=5).

The only antigen dose capable of inducing anti-CrylAc antibodies

in the small intestine fluid when administered by both routes was

100 j.kg (Fig 5). High IgG and low IgM intestinal antibody responses

were attained using the IP route, whereas moderate IgA and low IgM and IgG intestinal antibody responses were attained when the IG

route was used. The IgA antibody titers in the small intestine

fluid were higher than those of the other isotypes when sCrylAc was administered via IG.

1908 Immunogenicity of CrylAc Protoxin Vol. 64, No. 21, 1999

IgM

hG ,

IgA +

IgM

IgG k

@A

0.119

0.8 0,6 0.4 0,2 O-0 032 OS4 0.6 Ot8 A

492

Fig 5

Anti-CrylAc antibody secretions in small intestine induced

by several doses of CrylAc. The antigen (0.1, 1, 10, 100 pg

of CrylAc) was administered by IP or IG route. To collect

the intestinal contents, immune mice were sacrificed and

the small intestine flushed out with 5 ml of cold PBSM

buffer. The IgA, IgG and IgM coproantibody levels were

determined by ELISA. The A492 mean of control mice were

0.066 f 0.011. Bars represent the level of antibodies

expressed in arbitrary units of A,,, f standard deviation of

each experimental group (n=S).

Our results demonstrate that the CrylAc protoxin from B.

thuringiensis var kurstaki HD73 is highly immunogenic and capable

of inducing a mucosal immune response when administered via IG or

Discussion

Vol. 64, No. 21, 1999 Immunogenic&y of CrylAc Protoxin 1909

IP. The soluble form was more efficient than the crystalline form

in inducing circulating IgG antibodies. The serum IgG antibody

titers generated by sCrylAc administered via IP were higher than

those attained with CT. The anti-CrylAc intestinal antibody

response initially measured in feces clearly evidenced a mucosal

stimulation of the immune system by both protoxin forms.

The crystalline form of the protoxin appeared to induce a systemic

but not a local response when administered by IG route, whereas

via IP it induced a response of IgG and IgM antibodies similar to

that of sCryIAc. As far as we know, cCrylAc has a novel

immunological feature not present in other proteins. This protoxin

form induced systemic but not local IgA antibodies when applied

via IG. Generally, the oral application of an antigen induces

local IgA or combined local and serum IgA response, but not serum

response alone. However, these findings clearly show differences

in the immune response elicited by cCrylAc in the systemic and

mucosal immune systems and they may corroborate that there is a

dichotomy between them.

The differences in the immunological behavior of both CrylAc forms

are probably related with the differences in their biophysical

properties (17). To process proteins crystals, antigen presenting

cells (APC) require an initial solubilization step for partial

protein proteolysis to occur in the lysosomal compartments and Eor

the presentation of the peptides generated by the class II MHC

system (18). When cCrylAc is administered via IP, protoxin crystals are mostly taken up by macrophages and possibly by other

APC cells, which process the antigens in acidic lysosomal vesicles

(19). Solubilization of cCrylAc normally occurs at high pH values

(2), a fact that may hinder the proteolytic processing of crystals

necessary for releasing the immunogenic peptides.

Anti-tumoral properties of cCrylA protoxin have been implicated in

the enhanced overall immunity induced after IP administration (5).

This protein possesses a high molecular weight and after proteolytical processing it yields a fragment stable at extreme pH

values and resistant to further proteolisis, which possibly allows it to persist under unfavorable conditions. These characteristics

have been found for other highly immunogenic proteins. The known

affinity of CrylAc for biotinylated proteins may be also related

with its efficient uptake by APC cells (20). Specific Cry-binding

proteins in vertebrate cells have not been found, but homologous

polipeptides belonging to the aminopeptidase N (21) and cadherin

(22) protein families have been identified of higher animal cells.

Positive and negative effects of CrylAc on the anti-CT antibody

titers were observed regarding the immunization route. An enhance

of anti-CT IgG coproantibody response was observed when CT was co-

administered with sCrylAc via IP. Prasad et al. reported that

1910 Immunogenicity of CrylAc Protoxin Vol. 64, No. 21, 1999

cCrylA inoculated via IP enhances the immune response of rats

against sheep red blood cells (5). These data altogether suggest

that Cry proteins could have an adjuvant effect when co- administered with other antigens.

At the beginning of this study, CT was used as an adjuvant because

the immunogenicity of CrylAc via oral was unknown, however, it was

not necessary in the following studies. The adjuvant effect of CT

on the response against CrylAc was poor, being only observed when

it was co-administered with cCrylAc via IG. CT is the major oral

adjuvant described up to date. Despite these fact, Elson et al.

and others have been enable to stimulate sIgA responses to

ovalbumin when it is mixed and given orally with CT, so the

adjuvanticity of CT may not apply to all antigens (31,23). The

adjuvanticity of CT may relate to and depend on its

immunogenicity: the response against KLH given orally with CT to

H-2 congeneic mice, which are major respondents to CT, was

significantly higher than in strains that are low responders (16).

Although Balb/c mice have been used to study the CT properties

(301, this toxin shows a lower ability to act as an adjuvant in

this strain.

The dose-response experiment allowed us to get a more accurate

appreciation of sCrylAc immunogenicity. The highest anti-CrylAc

serum titers were attained with the highest dose tested.

Antibodies were detected in the small intestine fluid only in mice

immunized with 100 pg of sCrylAc. Surprisingly, the small intestine

fluid contained specific IgG antibodies when CrylAc was

administered by the IP route. In contrast, IG immunization with

the same doses induced higher anti-CrylAc IgA antibodies. No

correlation between serum and small intestine fluid antibody

levels was observed, which suggests that serum is not the source

of IgG coproantibodies and that perhaps IgG isotype production is

stimulated by sCrylAc in the gut-associated lymphoid tissue using

the IP route. The obtainment of IgG titers higher than those of

IgA is a feature of mucosal inflammatory diseases such as chronic

gastritis and Crohn's disease (25). However, toxicity studies

submitted to the US Environmental Protection Agency support that

B. thuringiensis containing Cry proteins and free of P-exotoxin do

not produce significant adverse effects on laboratory mice and

rats (3).

We detected high anti-CrylAc IgG antibody levels in the large

intestine fluid from mice receiving the antigen via IP. IgG antibody secretion in the large intestine and genital tract has

been reported for mice immunized with several antigens using

different routes (26,27), These antibodies are thought to gain

access to the mucosal surfaces by passive diffusion from blood.

Vol. 64, No. 21, 1999 Immunogenicity of CryL4c ProtoXin 1911

The IP route was more efficient than the IG route in triggering an

anti-CrylAc intestinal immune response. For other antigens, the IP

route has shown to be effective in the induction of both systemic

and mucosal immune responses (28) because the peritoneal cavity is

known as a significant source of plasma cells that are later found

in the mucosal tissues (29). Many authors have shown that

peritoneal and mucosal lymphocytes are similar in B cell surface

phenotype, maturation pattern and specific antibody repertories,

but their stimulation routes are different (24).

Cry proteins might therefore constitute a valuable tool in mucosal

immune studies, particularly on the dynamics of intestinal IgG

production and its role in intestinal immune protection against

infectious diseases. The data presented in this study support the

idea that CrylAc may be used as a carrier to introduce epitopes

either parenterally or locally into mucosal tissues, and it may be

an adjuvant capable of inducing appreciable changes in the

mammalian immune system.

Acknowledgements

Thanks to Lit. R. Pajon and Lit. D. Prieto for their ideas. This

work was partially supported by Conacyt Grants 0797-3453 PN and

5106-M9406.

References

1. J. PAYNE and L. KIM, 10 271-275 (1992).

2. H. H6FTE and H. WHITELEY, Microbial. Rev. 53 242- 255 (1989).

3. J.T. McCLINTOCK, C.R. SCHAFFER and R.D. SJOBLAD, Pestic. Sci.

45 95-105 (1995).

4. R.I. VAZQUEZ-PADRON, D. PRIETO, G. DE AL RIVA and G. SELMAN

HOUSEIN, J. Immunol. Meth. 196 33-39 (1996).

5. S.S.S.V. PRASAD and Y.I. SHETHNA, Indian J. Exp. Biol. 14 285-

290 (1976).

6. S.S.S.V. PRASAD and Y.I. SHETHNA, Biochim. Biophys. Res. Commun. 62 517-521 (1975).

7. G.E ROWE, Ph.D. thesis. University of Western Ontario London,

Ontario, Canada (1990).

8. A.Z GE, R.M. PFISTER, and D.H. DEAN, Gene. 93 49-54 (1990)

9. G.M.F. WATSON and N.H. MANN, J. Gen. Microb. 134 2559-2565 (1988).

10.U.K. LAEMMLI, Nature. 227 680-685 (1970).

ll.M. M. BRADFORD, Anal. Biochem. 72 248-254 (1976).

12.J.E. COLIGAN, A.M. KRUISBEEK, D.H. MARGULIES, E.T. SAEVACH,

and W. STRUBER, Current Protocols in Immunology, 1.2.0-2.1.0,

John Wiley & Sons, New York (1996).

13.T.E. KOERTGE and E. BUTLER, Scan. J. Immunol. 24 567-574 (1986).

1912 Immunogenicity of CrylAc Protoxin Vol. 64, No. 21, 1999

14.L. MORENO-FIERROS, M.A. DOMfNGUEZ and F. ENRfQUEZ, Exp. Parasitol. 80 541-549 (1995).

15.A.SIGARROA, Biometria y Disefio Experimental,708-740, Editorial

Pueblo y Education, Havana (1985).

16.C.0 ELSON, Infect. Immun. 60 2874-2879 (1992).

17.K.W NICKERSON, Biotechnology and Bioengineering. 22 1305-1333

(1980).

18.C.V. HARDING, F. LEYVA-COBIAN and E.R. UNANUE, Immunol. Rev.

106 77-114 (1988).

19.K.L. MCCOY and R.H. SCHWARTZ, Immunol. Rev. 106 129-145.

(1988).

2o.c. DU and K.W. NICKERSON, Appl. Environ. Microbial. 62 2932-

2939 (1993).

21.P.J.K. KNIGHT, N. CRICKMORE and D.J. ELLAR, Mol. Microbial. II

429-436 (1994).

22.R.K. VADLAMUDI, E. WEBER, I. JI, T.H. JI and L.A.Jr. BULLA. J.

Biol. Chem. 270 5490-5494 (1995).

23.A.D WILSON, C.J. CLARKE and C.R. STOKES, J. Immunol. 139 3764-

3772 (1990).

24.J. MESTECKI, R. ABRAHAM and P.L. OGRA, Handbook of Mucosal

Immunology, 357-371, Academic Press, New York .(1994).

25.H.C.REINEKER, S. SCHREIBER, W.F. STENSON and R.P. MACDERMOTT,

Handbook of Mucosal Immunology, 439-456, Academic Press, New

York (1994).

26.B. HANEBERG, D. KENDALL, H.M. AMERONGEN, F.M. APTER, J.P.

KRAEHENBUHL and M.R. NEUTRA, Infect. Immun. 62 15-23 (1994).

27.W.S. GALLICHAN and L. ROSENTHAL, Vaccine. 13 1589-1595 (1995).

28.S.M. MICHALEK, J.H. ELDRIDGE, R. CURTISS III and K.L.

ROSENTHAL, Handbook of Mucosal Immunology, 373-390, Academic

Press, New York (1994).

29.N. SOLVASON, A. LEHUEN and J.F. KEARNEY, Int. Immunnol. 3 543-

550 (1991).

30.G. HAJISHENGALLIS, S.K. HOLLINGSHEAD, T. KOGA and M.W.

RUSSELL, J. Immunol. 154 4322-4332 (1995).

31.c.o. ELSON and M.T. DERTZBAUGH, Handbook of Mucosal

Immunology, 391-402, Academic Press, New York (1994).